The two key requisites for successful gene therapy using hematopoietic stem cells (HSC) are gene transfer into a sufficient number of pluripotent HSC and gene expression at an appropriate level in the relevant mature hemato-lymphoid cells to achieve the therapeutic goal. This proposal is directed to the latter issue of gene expression from retroviral and lentiviral vectors after gene transfer into HSC. In the previous funding period, we have found that two multiply-modified retroviral LTRs, MND and MTD, showed significantly improved expression, compared to the standard Moloney Murine Leukemia virus (MLV) LTR, in murine ES cells and in murine hematopoietic and lymphoid cells. The greater frequency of active expression by the LTR of the modified vectors was accompanied by decreased methylation of cytosine residues in LTR. The mechanisms by which these modifications to the vectors augment expression are not clear, especially since the nature of changes made to the LTR are different. Incorporation of lineage-specific internal promoters within retroviral vectors may allow gene expression to be directed to specific blood cell types. Decreasing cis-inhibitory effects on internal promoters by use of our modified vectors may allow higher levels and/or fidelity of lineage-specific expression. Additionally, these modified LTR and internal promoters may be useful in lentiviral vectors, which show improved gene transfer into human HSC. To study these issues, we propose the following specific aims: 1. Analyze the mechanism of augmented expression produced by removal of the ncr (MND) or addition of the thy-1 fragment (MTD) or beta-IFN SAR by producing focused sets of mutations in the ncr and thy-1 sequences and analyzing effects on vector expression, 2. Characterize expression by retroviral and lentiviral vectors in primary murine hematopoietic and lymphoid cells derived from HSC in a gene transfer/BMT model, and 3. Characterize expression by retroviral and lentiviral vectors in primary human cells derived from transduced human CD34+ HSC using: a) cells derived in vitro using conditions of lineage-restricted cell differentiation (myeloid, erythroid, and B lymphoid), and b) cells derived in vivo using xenograft of human cells in immune-deficient mice (myeloid and T lymphoid). In all, these studies will produce new information concerning gene expression from retroviral and lentiviral vectors after gene transfer into HSC. More predictable, consistent expression is important to development of effective clinical gene therapy using hematopoietic stem cells.